Although aromatase inhibitors, such as letrozole, are the standard endocrine therapy of choice for post-menopausal women with early-stage metastatic estrogen-dependent breast cancer, the major limitation in managing this disease is the development of drug resistance. Although most letrozole-resistant breast tumors exploit and target growth factor receptor signaling, those tumors still express levels of the ER and this may play a more important role in cancer progression than is currently known. The long-term goal is to become an independent investigator focused on understanding the mechanism(s) of aromatase inhibitor-resistant breast cancer, in order to contribute to both the improvement of current therapeutic modalities and the development of novel treatment strategies. The objective of this application is to identify the anti-proliferative mechanism(s) of the phyto-antiestrogen, glyceollin I, on the growth of letrozole resistant breast cancer cells expressing low levels of ER. The central hypothesis of the proposed research is that glyceollin I has an anti-proliferative effect on letrozole-resistant breat cancer cells through inhibition of the expression of estrogen responsive genes, both in vitro and in vivo. The rationale for the proposed research is that identification of effective treatment strategies for postmenopausal breast cancer patients whose tumors relapse on letrozole treatment will result in both increased survival rates and enhance the quality of life among patients. The first specific aim is to determine the mechanism underlying the effects of glyceollin I inhibition of letrozole-resistant breast cancer cells. We will accomplish this aim by testing the hypothesis that glyceollin I decreases expression of ER-responsive genes critical for proliferation through inhibiting key components of ER-dependent signal transduction path- ways that contribute to cell proliferation, metastasis and tumorigenesis. We will utilize the following approach: letrozole-resistant breast cancer cells (LTLT-Ca) will serve as a model system to measure cell proliferative, estrogen responsive gene expression, cell motility and protein expression. The second aim is to identify the impact of glyceollin I treatment on letrozole-resistant tumor development in vivo. We hypothesize that the in vitro anti-proliferative effects of glyceollin I on LTLT-Ca cells will translate to decreases in tumor growth of letrozole-resistant tumors in vivo and this will be accomplished using the following approach: ovariectomized female nude mouse model implanted with LTLT-Ca cells will serve as a model to study the effect of glyceollin I on tumor growth and estrogen responsive gene expression. This contribution is significant because it will be the first critical step in the series of studies leaing to the development of novel phyto-antiestrogens used therapeutically in the treatment of letrozole refractory breast cancer. The proposed research is innovative, in our opinion, because ER-dependent signaling remains a crucial pathway in letrozole-resistant breast cancer, and glyceollin I, represents an estrogen-independent therapeutic that would be effective after initial hormone receptor-based therapy has failed.